Reverse genetics system for emerging tick-borne orthonairovirus highlights dispensable N-terminal region of Gn associated with replication in tick vectors

A growing number of emerging and re-emerging tick-borne orthonairoviruses belonging to the Sulina and Tamdy genogroups have recently been identified in association with febrile diseases in East Asia. Yezo virus (YEZV) is one such viruses and is genetically distinct from well-characterized Crimean-Congo hemorrhagic fever virus. Orthonairovirus glycoproteins (Gn and Gc) expressed from a single glycoprotein precursor (GPC) gene mediate interaction with host cells, yet the organization and functions of the YEZV glycoproteins remain largely undefined. To characterize YEZV glycoproteins in the context of infection, we developed a reverse genetics system that enables recovery of recombinant YEZV entirely from cloned cDNAs. The recombinant wild-type virus exhibited growth properties comparable to those of the parental isolate in vitro and maintained pathogenicity in vivo. Proteomic analysis of purified virus particles produced in mammalian cells showed that peptide coverage of GPC-derived products began at residue 69. We therefore used the reverse genetics system to examine the functional importance of the GPC subregion upstream of residue 69. A mutant with a deletion of GPC residues 28 to 68 (rYEZV GPC{Delta}28-68), which retained the predicted signal peptide, was successfully recovered, suggesting that this subregion is dispensable for producing infectious virus. rYEZV GPC{Delta}28-68 propagated at levels comparable to those of the wild-type virus in mammalian cells and exhibited similar pathogenicity in a mouse model. Interestingly, the mutant reached lower viral titers in tick-derived ISE6 cells and in ticks in vivo, suggesting that this N-terminal subregion may contribute to virus replication in ticks. Together, these findings demonstrate the utility of the newly established reverse genetics system for dissecting the functions of the YEZV glycoproteins in mammalian and tick systems.